The present invention relates to protein hydrolysates and their use in foodstuffs.
Hydrolysed proteins from a variety of sources are used widely in the food industry. For instance, they are commonly employed as a component in dehydrated soups, as flavourings and in other processed foodstuffs to obtain e.g. food flavourings after a Maillard reaction. They also find medical use as dietary supplements for patients suffering from a variety of diseases and metabolic disorders. Relatively new developments are their use in products for consumers with non-medical needs as athletes or people on a slimming diet and in personal care applications. Furthermore in situ hydrolysed proteins play an important role in the development of flavours in fermented food products. In the latter products the microbial starter cultures used usually excrete proteolytic enzymes responsible for hydrolysis of the raw material into amino acids. Metabolic transformation of these aminoacids leads to potent flavour compounds and volatiles characteristic for e.g. fermented dairy products such as cheese or yogurts, various meat products, beers and wines.
Although conventional protein hydrolysates are prepared by subjecting the protein source to harsh chemical conditions, there has been an increased interest in obtaining such hydrolysates by enzymatic hydrolysis. Both the chemical and the enzymatic route aim to release high levels of amino acids from the protein source with maximum efficiency and lowest cost. For that reason cheaply available protein sources like soy meal and wheat gluten are popular substrates for preparing hydrolysates. To liberate as many amino acids as possible, the enzymatic route employs either complex mixtures of several endo- and exoproteases (e.g. International Patent Application WO94/25580) or it combines endoproteases with a single but broad spectrum exoprotease (International Patent Application WO-A-98/27827). In all cases the aim is to obtain a high degree of hydrolysis and an end product that contains a large variety of free amino acids.
Though free amino acids as such, can elicit a number of taste impressions, these taste impressions are very basic (bitter, sweet, sour and “umami”) and the amino acid concentration required for perceiving these tastes are high. Threshold values for individual amino acids can range from 0.3-80 millimoles/liter. Despite these high threshold values, free amino acids can create major sensory effects at much lower concentration ranges through a number of mechanisms.
One of these mechanisms involves free glutamate and can create strong savoury enhancing effects because of the synergy between glutamate and 5′-ribonucleotides. If combined with proper concentrations of 5′-ribonucleotides such as 5′-IMP and 5′-GMP, the detection threshold of the umami taste generated by glutamate is known to be lowered by almost two orders of magnitude.
Another flavour enhancing mechanism involves Maillard reactions. Compared with free amino acids, Maillard products in which free amino acids have been reacted with sugars exhibit much more impressive taste and odour characteristics. In Maillard reactions overwhelmingly complex flavour and odour systems can develop with threshold values that are several orders of magnitude lower than those recorded for the free amino acids.
Maillard products are formed at elevated temperatures usually during cooking, baking or roasting when preparing food. During these treatments both colour and a large array of aromas develop. In these reactions amino groups react with reducing compounds as a first step and ultimately leading to a whole family of reaction pathways. In foods the amino compounds involved are predominantly free amino acids and proteins and the reducing compounds primarily represent reducing sugars. Factors that influence the Maillard reaction include the type of sugar and amino acid involved as well as physical factors such as the pH, temperature, water activity (aw), reaction time, and so on.
Both mono as well as disaccharides can take part in the Maillard reaction. Generally speaking aldoses are more reactive than ketoses and pentoses more than hexoses or disaccharides, and so whereas the type of sugar strongly influences the amount of flavouring compounds generated, the amino acid involved in the reaction largely determines the nature of the flavour formed. For example, the inclusion of pure methionine in Maillard reaction systems often leads to vegetable or stewed notes, pure cysteine leads to meat-like flavours, pure proline, hydroxy proline and leucine to bakery aromas (R. F. Hurrell, Food Flavours, Part A: Introduction, Elsevier Scientific Publishing Company, Eds.: I. D. Morton and A. J. Macleod). Since these results have been obtained using pure amino acids rather than mixtures of several amino acids, as occur in food ingredients, it is evident that the outcome represents only a gross simplification of the natural situation. Likewise, the sugars that naturally occur in food will have an impact, and further complicate and affect the development of taste and aroma.
Apart from Maillard reactions, amino acids can also undergo important chemical transitions at ambient temperatures. The latter type of transitions are enzyme dependent and are quite common in fermented foods such as beer, yogurt, cheese ripening and meat and wine maturation processes. In these fermentation processes, free amino acids are liberated from the raw materials used by proteolytic enzyme activity from either the raw material or the microbial starters used. During the maturation phase microbial metabolic activity then converts the free amino acids into derivatives with increased sensoric properties. For example, L-leucine, L-isoleucine and L-valine lead to the formation of valuable fusel alcohols like amylalcohols and isobutanol in beer fermentation. L-leucine is known as the precursor for cured meat compounds such as 3-methylbutanal and 3-methylbutanol, whereas L-phenylalanine can lead to benzacetaldehyde. Similarly cheese volatiles such as methanethiol and dimethyldisulphide have been traced back to the occurrence of methionine in cheese as well as methylpropanoic acid and methylpropanal to valine. Accordingly the “sur-lie” method used in wine making and known to generate tastier wines, can be ascribed to the increased presence of amino acids such as aspartic acid, arginine, alanine, leucine and lysine.
Prior art processes for protein hydrolysis (WO94/25580 and WO98/27827) aim at releasing all available amino acids and the presence of so many different amino acids will blur the desired pronounced taste or aroma note in the final product.
WO98/14599 refers to certain polypeptides obtained from Aspergillus oryzae and to hydrolysates prepared with these polypeptides in combination with (specific or unspecific) endopeptidases and (specific or unspecific) exopeptidases. WO98/14599 mentions hydrolysates that have an increased content of Leu, Gly, Ala and/or Pro, such as 1.1 times greater but uses for such hydrolysates are not mentioned.
European patent application EP-A-799577, describes a whey protein hydrolysate wherein the Phe (phenylalanine) content is reduced. This whey protein hydrolysate is used as food for patients suffering from PKU (phenylketonuria).
Voigt et al (Food Chemistry 51 (1994) pp. 7-14) describes the production of the cocoa-specific aroma precursors by in vitro proteolysis of seed proteins. Cocoa-specific aroma precursors can only be obtained by specific hydrolysis of only one substrate, which is cocoa vicillin-class globulin proteins.